The conserved dinucleotide AG of the 3' splice site may be recognized twice during in vitro splicing of mammalian mRNA precursors.

We have previously used site-directed mutagenesis to introduce an additional branch site into the first intron of the human beta-globin gene at nt -24 between the natural branch site (nt -37) and the normal 3' splice site (nt -1). We found that either the upstream or downstream branch site could be used during in vitro splicing, depending on which site best matched the mammalian branch site consensus YURAC (R = purine; Y = pyrimidine). Here we show that introduction of an additional AG dinucleotide at nt -20 between the downstream branch site and the normal 3' splice site results in alternative 3' splicing. Splicing to the new AG uses the upstream branch site exclusively, presumably because the downstream branch site is only 4 nt from this 3' splice site. We were surprised, however, to find that the presence of the new AG also prevents the use of the upstream branch site for splicing to the normal 3' splice site. Analysis of additional mutants confirmed earlier work [Krainer et al.: Mechanisms of human beta-globin pre-mRNA splicing. In Berg, P. (Ed.), The Robert A. Welch Foundation Conferences on Chemical Research XXIX. Genetic Chemistry: The Molecular Basis of Heredity. Welch Foundation, Houston, TX, 1985, pp. 353-382] that the new AG cannot function by itself as a complete 3' splice site; rather, it appears that alternative 3' splicing initiates at the normal 3' splice site but then searches, once the reaction is underway, for the first AG downstream from the chosen branch site. Taken together, our data suggest that the conserved AG dinucleotide at the 3' splice site may be recognized twice during mammalian mRNA splicing in vitro.